Lelystad Biologicals BV

Lelystad, Netherlands

Lelystad Biologicals BV

Lelystad, Netherlands
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van Wielink R.,Central Veterinary Institute of Wageningen UR CVI | van Wielink R.,Wageningen University | Kant-Eenbergen H.C.M.,Central Veterinary Institute of Wageningen UR CVI | Harmsen M.M.,Central Veterinary Institute of Wageningen UR CVI | And 3 more authors.
Journal of Virological Methods | Year: 2011

Madin-Darby canine kidney (MDCK) cells are currently considered for influenza vaccine manufacturing. A drawback of these cells is their anchorage dependent growth, which greatly complicates process scale-up. In this paper a novel MDCK cell line (MDCK-SFS) is described that grows efficiently in suspension and retained high expression levels of both α-2,6 and α-2,3 sialic acid receptors, which bind preferably to human and avian influenza viruses, respectively. The production of avian influenza virus by BHK21, Vero and MDCK-SFS cell lines was compared. Although BHK21 cells consisted of two populations, one of which lacks the α-2,3 receptor, they supported the replication of two influenza strains to high titres. However, BHK21 cells are generally not applicable for influenza production since they supported the replication of six further strains poorly. MDCK-SFS cells yielded the highest infectious virus titres and virus genome equivalent concentration for five of the eight influenza strains analyzed and the highest hemagglutination activity for all eight virus strains. Taken together with their suitability for suspension growth this makes the MDCK-SFS cell line potentially useful for large scale influenza virus production. © 2010 Elsevier B.V.


Harmsen M.M.,Central Veterinary Institute of Wageningen UR | Fijten H.P.D.,Central Veterinary Institute of Wageningen UR | Westra D.F.,Lelystad Biologicals BV | Coco-Martin J.M.,Lelystad Biologicals BV
Vaccine | Year: 2011

Intact (146S) foot-and-mouth disease virions (FMDVs) can dissociate into specific (12S) viral capsid degradation products. Using two single-domain antibody fragments that bind specifically to either 146S or 12S particles we developed two ELISAs for the quantification of these particles in FMDV antigen preparations used for vaccine manufacturing. Only O serotype strains are detected in the 146S specific ELISA whereas strains of most serotypes are detected in the 12S specific ELISA. However, the 146S concentration of A and Asia 1 serotype strains could be measured indirectly using the 12S specific ELISA by prior conversion of 146S into 12S particles by heat treatment. This allowed us to demonstrate that addition of the preservative thiomersal to FMDV antigens stimulates the dissociation into 12S particles of O, A and Asia 1 serotype strains upon prolonged storage at 4°C. FMDV dissociation is known to result in a strongly reduced immunogenicity, which was experimentally confirmed here. Therefore, we recommend to omit thiomersal from FMD vaccines to increase its shelf life. © 2011 Elsevier Ltd.


Harmsen M.M.,Lelystad Biologicals BV | Harmsen M.M.,Central Veterinary Institute of Wageningen UR | Jansen J.,Lelystad Biologicals BV | Westra D.F.,Lelystad Biologicals BV | Coco-Martin J.M.,Lelystad Biologicals BV
Vaccine | Year: 2010

We have used a novel method, surface-enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS), to characterize foot-and-mouth disease virus (FMDV) vaccine antigens. Using specific capture with FMDV binding recombinant antibody fragments and tryptic digestion of FMDV antigens the spectral peaks representing the FMDV structural proteins VP1, VP2, VP3 and VP4 were identified. VP1 existed as 2 variants differing by 0.2 kDa and VP4 as 8 variants differing by 14-17 Da. Such heterogeneities have not been reported earlier. They could represent oxidation of VP4 and N-glycation of VP1. We also detected FMDV proteolysis upon incubation at elevated temperatures and impurities in FMDV antigen preparations. Finally, we could also characterize FMDV antigen present in emulsions with oil adjuvant by SELDI-TOF-MS. Such FMDV antigen retained the VP4 protein which is known to be specifically present in intact (146S) FMDV particles but absent from specific (12S) degradation products. This indicates that virions do not dissociate upon emulsification. © 2010 Elsevier Ltd. All rights reserved.

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